LYMPHOID CELLS CONVERTED BY LYMPHOID RNA EXTRACTS IN VITRO AND IN VIVO TO SYNTHESIZE ALLOGENEIC...

LYMPHOID CELLS CONVERTED BY LYMPHOID RNA EXTRACTS IN VITRO AND IN VZVO TO SYNTHESIZE

ALLOGENEIC IMMUNOGLOBULINS * t

Clara Bell and Sheldon Dray

Department of Microbiology University of Illinois at the Medical Center

Chicago, Illinois 60680

INTRODUCTION

An immunologically active ribonucleic acid (RNA) t extract can be obtained from lymphoid cells immunized in vitro and can convert lymphoid fragments or cells from nonimmunized animals in vitro or in vivo to antibody-forming cell^.^-^ Fishman and colleagues extracted RNA from peritoneal exudate cells (PEC) obtained from nonimmunized rats or rabbits immunized to T2 phage in vitro and used neutralization of T, phage as the assay for antibody detection. Cohen and colleagues 5-7 and Friedman extracted RNA from the spleen of mice immunized with sheep red blood cells (SRBC) , and used the Jerne plaque technique for the assay of antibody. In these experiments 2-6. the RNA extracts were inactivated by ribonuclease (RNase) but not by deoxyribonuclease (DNase) or trypsin, which suggests that there is a requirement for intact RNA. The ineffectiveness of trypsin on the activity of RNA extracts did not rule out a role for small amounts of antigen present in the RNA extracts.10.12-14 In- deed, it has been shown that RNA-antigen complexes are more immunogenic than antigen alone and may act as superantigens.12. l5 The RNA may act as a nonspecific adjuvant or as a metabolic stimulator of antibody formation.1°18 Nevertheless, an informational role for the RNA is suggested by the experiments of Adler and colleague^,^ in which it was demonstrated that the IgM antibody first formed possessed the genetic markers (that is, the b locus allo- typic specificity) of the RNA donor rather than of the converted cells.

We have explored further the informational or possibly regulatory effect of immunologically active RNA extracts by the use of the genetic markers, namely, that is, allotypes, for light and heavy chains of rabbit immunoglobu-

* This study was supported in part by the National Institute of Allergy and Infec-

t This paper was awarded the 1972 Boris Pregel Award for Research in Biology. t Abbreviations: SRBCZsheep red blood cells; IgG=immunoglobulin G; IgM

=immunoglobulin M; RNase=ribonuclease; DNase=deoxyribonuclease; RNA= ribonucleic acid; HBSSzHanks’ balanced salt solution; DEAE=diethylaminoethyl; 5-day i-RNA or 5-day i-SpC=RNA or SpC from a rabbit 5days after a single injection of SRBC; 24-day i-RNA or 24-day i-SpC=RNA or SpC from a rabbit 24 days after multiple injections of SRBC; ni-RNA or ni-SpC=RNA or SpC from nonimmunized rabbits; anti-IgG-Fc=goat antibody specific for the Fc-fragment of IgG; b4 and bS=allelic allotypes of kappa light chains; a l , a2, and a3=allelic allotypes of the variable region of the heavy chains; b4 RNA, b5 SpC, alb4 RNA, a3b5 SpC, and so on, denote the allotypes of homozygous rabbits from which the RNA and SpC were obtained.

200

tious Diseases research grants AI-07043 and AI-10138.

Bell & Dray: Lymphoid Cells 20 1

lins.l”--?S In this paper we present the overall results of our work to date and discuss its implications.

The methods used in our studies have been described in detail and are as follows: (1) the SRBC were used as the antigen; (2) the localized hemolysis in gel technique was used to assess the number of IgM, “direct” and I@, “indirect” plaque-forming cells; 11, 19- 200. 27 (3) radioautography of the plaques with antiallotype antibodies was used to identify the allotype in the plaques; 10. ? ( I , ?2. 2 1 (4) inhibition of plaque formation with antiallotype antibodies was also used to identify the allotype in the plaques; lo, -?O ( 5 ) immunodiffusion was used to identify the allotype of the IgG in lymphoid cell lysates or serum; Z 1 - 2 2“ (6) the localized hemolysis in gel technique was used to assess the IgM and IgG anti-SRBC antibody titers in lymphoid cell lysates or serum; 20* L‘D and (7) the hot phenol method was used to obtain RNA extracts from lymphoid tissues.lO- 3 1 ) v 31 The RNA extracts were obtained from lymphoid tissues (spleen and lymph nodes) of nonimmunized rabbits (ni- RNA), at the peak of the IgM antibody response (5day i-RNA), and at the peak of the IgG antibody response (24-day i-RNA) (FIGURE 1). Rabbits homozygous for the kappa light-chain allotypes, b4 or b5, and/or for the heavy-chain allotypes, a l , a2, or a3, were used in all of the experiments.

R N A Conversion of Spleen Cells in Vitro

Direct and Indirect Plaques

Spleen cells of nonimmunized rabbits (ni-SpC, 2 X lo7 in 0.1 ml HBSS) were incubated for 15-30 min at 37” C with 5 d a y I-RNA (45-700 pg in 0.1 ml HBSS) or 24-day i-RNA (150-1000 pg in 0.1 ml HBSS) respectively (FIGURE 2) . After the cells had been washed with HBSS to remove the RNA in the supernatant fluid, the cell pellet was resuspended in HBSS and plated with SRBC in agarose. The number of IgM direct and IgG indirect PFC/lOs viable SpC were determined by scanning the plates 4 and 18 hours later (FIGURE 2) . The “direct” plaques were developed with complement (FIGURE 2) ; they were inhibited by the presence of 2-mercaptoethanol (2-ME) or anti-IgM but not by anti-IgG.1” The indirect plaques were developed with anti-IgG and the subsequent addition of complement (FIGURE 2) ; they were inhibited by the presence of excess anti-IgG but not by 2-ME or anti-IgM.Zo

In 39 selected experiments, the 5-day i-RNA of 39 immunized rabbits successfully converted the SpC of 30 nonimmunized rabbits (ni-SpC + 5-day i-RNA, FIGURE 3) to yield an average of 287 direct PFC (range: 130-375). This response was similar to the average of 314 PFC (range 220-382) which was observed for 21 SpC suspensions obtained 5 days after rabbits had received a single injection of SRBC (5-day i-SpC, FIGURE 3) . The RNA extracted from 12 nonimmunized animals (ni-RNA) was ineffective, yielding an average of only 5 PFC (range: 1-7) which is the background level found with ni-SpC (range: 2-9) of 30 nonimmunized rabbits (FIGURE 3).

In 36 selected experiments, the 24-day i-RNA of 36 immunized rabbits successfully converted ni-SpC to yield an average of 268 indirect PFC (range: 109-537). This was about half of the average response of 521 PFC (range:

202 Annals New York Academy of Sciences

t FIOURE 1. Hemolysin response curve in rabbit serum at various times: (1) after

one intravenous injection of 4-8x 108 SRBC, that is, direct IgM hemolysin (first peak); (2) after several (2-10) intravenous injections of 4 4 x 1 0 8 SRBC, that is, enhanced indirect IgG hemolysin (second peak). Each point at 0-4, 8-16, and 28-32 days represents the mean determination of the hemolysin response curves from serum of 5 rabbits, whereas each point at 5-7 days and 18-24 days represents the mean determination of hemolysin from serum of 12 rabbits. Direct IgM hemolysin and enhanced indirect IgG hemolysin were determined by the methods of Uyeki and KIassen,= as described elsewhere.2o

368-721) observed for 18 SpC suspensions obtained 2 to 3 days after the last of the multiple injections of SRBC (24-day i-SpC) (FIGURE 3). The RNA extracted from 10 nonimmunized animals (ni-RNA) was ineffective, yielding an average of only 4 PFC (range: 0-8) which is the background level found with the ni-SpC (range: 0-8) of 26 nonimmunized rabbits (FIGURE 3).

The data presented above represent approximately 40% of the total experiments. The remaining experiments were not successful in yielding suffi- ciently large numbers of plaques and were discarded. Contamination of the system with RNase, the presence of phenol in the RNA extracts, and unrespon- sive rabbits probably account for most of the unsuccessful experiments.

Bell & Dray: Lymphoid Cells 203

The R N A Extracts

The amount of RNA that is maximally effective in the conversion of ni-SpC varies with each RNA preparation. Each RNA preparation was therefore assessed at several different concentrations in preliminary experiments in order to select an optimal concentration. In our earlier experiments,*0. 2o we had reported that approximately 500 pg 5day i-RNA and 1000 pg 24-day i-RNA yielded maximal responses. More recently, however, relatively large numbers of PFC have been obtained by conversion with as little as 45 pg 5-day i-RNA and 150 pg 24-day i-RNA. The RNA preparations were inactivated by RNase but not by DNase or trypsin (TABLE t ) . l D * ?" The sucrose density gradient pattern was essentially unchanged after treatment with DNase or trypsin, but with

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FIGURE 2. Schematic representation of the experiments employed to assess the conversion of NI-SpC in vifro by RNA extracts to yield direct and indirect plaque- forming cells (PFC). The ni-SpC (2x10' in 0.1 ml HBSS) were incubated for 15- 30 minutes at 37°C with 5-day I-RNA (100-700 p g in 0.1 ml HBSS) or with 24day i-RNA (150-1,000 ag in 0.1 ml HBSS). We removed the RNA from the supernatant fluid by washing the cells with 100 volumes of HBSS. The RNA-treated SpC were resuspended in HBSS and plated with 10% SRBC in agarose. The plates were incubated at 37°C for 1-2 hours, prior to the addition of a 1:lO dilution of guinea pig complement in order to develop the IgM direct plaques. To develop the IgG indirect plaques, anti-IgG-Fc was added prior to the addition of complement. The direct and indirect plaques were scanned after an additional 18 hours at 4"C.'@*


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FIGURE 3. Direct and indirect p1aque:forming cells (PFC/lV SpC) among spleen cells obtained as indicated. Each vertical bar shows the results of several experiments. For direct PFC, the numbers of experiments were 21 for 5-day i-SpC, 30 for ni-SpC, 39 for niSpC+S-day 1-RNA, and 12 for ni-SpC+ni-RNA. For indirect PFC, the numbers of experiments were 18 for 24-day iSpC, 26 for ni-SpC, 36 for ni-SpCf24- day i-RNA, and 10 for niSpC+ni-RNA. Each experiment was done with a different combination of rabbits as a source of RNA and SpC. The arrow indicates the mean value; the shaded area indicates the range of values.

RNase larger numbers of molecules of lower molecular weight were observed. These results imply that intact RNA is necessary for activity.

Active Synthesis of Anti-SRBC Antibodies

The active synthesis of anti-SRBC antibodies by i-RNA-converted ni-SpC was evaluated by assaying the anti-SRBC titer in RNA extracts, lysed preparations of ni-SpC, and lysed preparations of the same number of ni-SpC which had been preincubated for 95 min with the same amount of i-RNA (FIGURE 4). The IgM anti-SRBC titer obtained when ni-SpC were incubated with 5-day i-RNA was 1:1024; the IgG anti-SRBC titer obtained when ni-SpC were incubated


with 24-day I-RNA was 1:4096 (FIGURE 4 ) . These values are substantially higher than those obtained with ni-RNA or untreated ni-SpC. Furthermore, no anti-SRBC activity was detected in the RNA extracts. To assess the possibility that preformed anti-SRBC antibody may be present in an undetectable combination with the RNA, each RNA preparation was pretreated with RNase and when the above experiments were repeated, only background titers were observed.

The Allotypes in the Antibody Plaques

The light- and heavy-chain allotypes of the IgM direct plaques were determined by radioautography and by the inhibition of plaque formation with antiallotype antibodies. The results of two typical experiments are presented: a2b5, 5-day i-RNA + alb4 ni-SpC (FIGURE 5 ) and alb4, 5-day i-RNA + a3b5 ni-SpC (FIGURE 6 ) . By radioautography, the RNA donor's light-chain allotype was present in 81-90% of the plaques; the RNA donor's heavy-chain allotype was present in 75-76% of the plaques. The host allotypes, light and heavy, accounted for only 3-6% and 6 1 0 % of the plaques respectively.

The light- and heavy-chain allotypes of the IgG indirect plaques were also identified by using antiallotype antibodies (either by inhibiting plaque formation or by developing radioplaques). The results of two typical experiments are presented: alb5, 24-day I-RNA + a3b4 ni-SpC (FIGURE 7 ) and a2b4, 24-day i-RNA + alb5 ni-SpC (FIGURE 8) . By radioautography, the RNA donor's light- chain allotype was present in 74-79% of the plaques; the RNA donor's heavy- chain allotype was present in 55-59% of the plaques. The host allotypes, light and heavy, accounted for 15-17% and 2 6 2 5 % of the plaques respectively.

In all of these experiments, the results obtained by radioautography agreed

TABLE 1 EFFECT OF RNASE, DNASE, AND TRYPSIN ON THE CONVERSION OF NI-SPC TO DIRECT AND INDIRECT PFC BY TEN PREPARATIONS OF 5-DAY I-RNA AND OF 24-DAY I-RNA * ~ _ _ _ _

Total PFC/lO" SpC Percentage of Total PFC/lOs SpC after RNase DNase Trypsin

mean range mean range mean range mean range

Direct PFC 330 250-372 5 0-20 95 91-98 91 84-96 Indirect PFC 275 140-392 12 3-21 95 90-99 93 89-96

* 10-40 pg RNase/O.l ml HBSS were incubated with 500 pg 5-day immune RNA/ 0.1 ml HBSS, or 500-1,000 pg 24-day immune RNAlO.1 ml HBSS, for 10 min at 26°C. 10-20 pg DNad0 . l HBSS and 10 pg trypsin/O.l ml HBSS were incubated with 500 pg 5-day immune RNA/O.I ml HBSS or with 500-1,000 pg 24-day immune RNA/O.l ml HBSS respectively for 30 min at 37°C. The RNA was reextracted with phenol and reprecipitated with alcohol. Then 2 X 10' SpC derived from nonimmunized rabbits (in 0.1 ml HBSS) were added to the enzyme-treated RNA preparations and incubated for an additional 15 min at 37°C. The supernatant fluid was removed and the cells were washed with HBSS before being assayed for direct and indirect PFC.


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L/ FIGURE 4. Evidence for the active synthesis of IgM and IgG anti-SRBC by RNA-

converted SpC." The mixtures in the tubes contained ni-SpC (0.9x 10' SpC/ml) and/or i-RNA, as indicated, in a total volume of 0.2 ml HBSS. The mixtures were incubated for 15 minutes at 37°C. 'The volume was increased to 0.5 ml HBSS; incubation at 37°C was continued for another 80 min and was followed by 10 cycles of freezing and thawing. The lysates were concentrated by ultrafiltration at 4°C to 100 pl. The antiSRBC hemolysin direct titer and enhanced indirect titer (that is, total anti-SRBC titer using goat anti-IgG-Fc) were determined by the localized hemolysin- in-gel methods of Uyeki and KlassenPB as described elsewhere."

closely with those obtained by the inhibition of plaque formation, the latter method yielding slightly higher results. Most of the plaques possessed the light- and heavy-chain allotypes of the RNA donor. The results imply that at least two kinds of RNA molecules are functioning, one for the light chain and one for the heavy chain. Differences in the optimal amounts of these two RNA molecules may account for the somewhat lower efficiency of conversion with respect to the heavy-chain allotypes.

Bell & Dray: Lymphoid Cells

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Active Synthesis of the R N A Donor's IgG Allolypes

The allotypes of the IgG in the lysates of SpC mixtures incubated for 30 min in RNA and for an additional 4 hours in the presence of SRBC were assayed by immunodiff usion. In each experiment these lysates exhibited the RNA donor's light- and heavy-chain allotypes (FIGURE 9, left column: I. well 4 in the upper two diagrams; 11. well 8 in the lower two diagrams). Moreover,

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~-IGURES 5 AND 6. Percentage of IgM direct plaques that bear the KNA donor and SpC host light- and heavy-chain allotypes among ni-SpC treated in vitro with S-day I-RNA extracts. The two 5-day I-RNA preparations (extracted from spleen and lymph nodes) were obtained from a2b5 (FIGURE 5 ) and alb4 (FIGURE 6) doubly homozygous rabbits, 5 days after a single intravenous injection of 4>( 108 SRBC. The host ni-SpC were obtained from alb4 (FIGURE 5) and a3b5 (FIGURE 6) doubly homozygous nonimmunized rabbits. The host ni-SpC ( 2 . 2 ~ 10' in 0.1 ml, FIGURE 5; 2 . 0 ~ lo' in 0.1 ml, FIGURE 6) were incubated with the 5-day i-RNA (430 p g in 0.1 ml, FIGURE 5; 530 pg in 0.1 ml, FIGURE 6) for 15 minutes at 37°C and were washed prior to plating. The allotypes of the b kappa light-chain locus and the a heavy-chain locus for the IgM antibodies in the direct plaques were identified (1) by inhibition of plaque formation in the presence of antiallotype antibodies incorporated into the agarose mixture (clear bars), and (2) by radioautography with IgG isolated from anti-b4, anti-b5, anti-al, anti-a2, and anti-a3 antisera and labeled with =I (shaded bars). Each vertical bar shows the average of quadruplicate determinations. All experiments in FIGURES 5 or 6 were done with a single SpC population obtained from one rabbit and treated with a single RNA preparation.


well 4 was filled with essentially the same amount of a2b5 RNA as well 2 and with the same amount of lysed alb4 SpC as well 3 (FIGURE 9, left upper two diagrams). Therefore, the precipitin line which appears from well 4 indicates

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FIGURES 7 and 8. Percentage of IgG indirect plaques that bear the RNA donor and SpC host light- and heavy-chain allotypes among ni-SpC treated in vitro with 24- day i-RNA extracts. The two 24-day i-RNA extracts (from spleen and lymph nodes) were obtained from alb5 (FIGURE 7) and a2b4 (FIGURE 8) doubly homozygous rabbits, 24 days after the initial intravenous injection and 2-3 days after the last of 7 injections of 8x loR SRBC. The host ni-SpC were obtained from a3b4 (FIGURE 7) and alb5 (FIGURE 8) doubly homozygous nonimmunized rabbits. The host ni-SpC ( 2 . 0 ~ 10' in 0.1 ml, FIGURE 7; 2.3 x 10' in 0.1 ml, FIGURE 8) were incubated with 24- day i-RNA (580 pg in 0.1 ml, FIGURE 7; 720 pg in 0.1 ml, FIGURE 8) for 15 minutes at 37°C and were washed prior to plating. The allotypes of the b kappa light-chain locus and the a heavy-chain locus for the IgG antibodies in the indirect plaques were identified (1 ) by inhibition of plaque formation in the presence of antiallotype antibodies incorporated into the agarose (clear bars), and (2) by radioautography with IgG isolated from anti-b4, anti-b5, anti-al, anti-a2, and anti-a3 antisera and labeled with T (shaded bars). Each vertical bar shows the average of quadruplicate determinations. All experiments in FIGURES 7 or 8 were done with a single SpC population obtained from one rabbit and treated with a single RNA preparation.

that the RNA-treated SpC had actively synthesized the allelic b5 and a2 allotypes of IgG. Also, well 8 was filled with essentially the same amount of alb4 R N A as well 6 and with the same amount of lysed a3b5 SpC as well 7 (FIGURE 9, left lower two diagrams), Again, the precipitin line which appears


FIGURE 9. Identification of kappa light-chain allotypes (b4 and b5) and heavy- chain allotypes (a l , a2, and a3) as well as IgM and IgG in SpC lysates by double diffusion in 1.0% agarose gel containing 0.75% deoxycholate in 0.1 M sodium borate buffer at pH 8.6. The antibody reagents are in the center wells. The reference


from well 8 indicates that the RNA-treated SpC had actively synthesized the allelic b4 and a1 allotypes of IgG. The precipitin bands are approximately the same distance from the center antibody well (as the band from well 41, or if anything closer to the antibody well (as the band from well 8), than the coalescing band from the wells containing the 1 : 30 dilution of reference sera ( * I . This indicates that the concentration of the IgG allotype in the lysate of 2.2 X lo* cells in well 4 or 2.3 x lox cells in well 8 is of the same order of magnitude as the concentration of the allotype in a 1:30 dilution of whole serum, and this is approximately 0.3 mg/ml. Since approximately 25 pl reference serum was added to each lateral well, the amount of IgG obtained from 2.2 X loR or 2.3 x lox cells was approximately 7-10 pg.3z The lysates of untreated SpC exhibited only the host allotypes and the RNA treatment left this reaction intact (FIGURE 9, middle column: I. wells 3 and 4; 11. wells 7 and 8). Both IgG and IgM were found in the lysates of the untreated SpC and the RNA-treated SpC (right column: I. wells 3 and 4; 11. wells 7 and 8) . None of the antibodies detected IgG or IgM in the RNA preparations (wells 1 and 2; wells 5 and 6). The quantitative considerations of this immunodiffusion experiment clearly indicate that the RNA-treated SpC actively synthesize the allelic allotypes of the RNA donor.

RNA CONVERSION OF SPLEEN CELLS in Vivo

Injection of RNA-Treated Peritoneal Exudate Cells

Peritoneal exudate cells of nonimmunized rabbits ( 1.7-2.5 X lo8 ni-PEC) were incubated with 5 d a y I-RNA for 30 min and for an additional 4 hours with SRBC (lo*) and then were reinjected intravenously into the same rabbit. The spleens were assayed 5 days later for IgM direct plaques having the RNA donor and SpC host's light-chain allotype (FIGURE 10). By radioautography, 32% of the plaques were of the donor's allotype in the first experiment (b5 RNA+b4 SpC) and 19% of the plaques were of the donor allotype in the second experiment (b4 RNA+b5 SpC) . In the control experiment (b4 RNA+b4 SpC), practically all the plaques were of the b4 allotype (FIGURE 10). In another set of experiments, the rabbits received two booster

reagents (*) are normal rabbit sera which possess the appropriate antigen: b4 for anti-b4, b5 for anti-b5, a1 for anti-al, a2 for anti-a2, a3 for anti-a3; normal rabbit sera (NRS) for anti-IgM and anti-IgC. The numbered wells contained the following reagents: (1) alb4 RNA (470 p g ) ; (2) a2b5 RNA (450 p g ) ; (3) alb4 SpC (2.2 x lo8 ni-SpC of rabbit A29); (4) a2b5 RNA (450 pg) +alb4 SpC $ 2 . 2 ~ lo" ni-SpC of rabbit A29); ( 5 ) a3b5 RNA (760 pg); (6) alb4 RNA (725 p g ) ; (7) a3b5 SpC (2 .3~108 ni-SpC of rabbit A35); (8) alb4 RNA (725 pg)+a3bS SpC (2 .3~10 ' SpC of rabbit A35). Each well that contained SpC (wells 3, 4, 7, and 8) was filled with the concentrated lysates pooled from ten incubation mixtures which contained one-tenth of the amounts listed, originally in a volume of 0.2 ml HBSS for 30 minutes at 37"C, and then in a volume of 25 ml HBSS containing 10' SRBC for an additional 4 hours, in a 37°C Con incubator, with periodic shaking before pooling and lysis by ten cycles of freezing and thawing. The 250 ml of lysate for each well was concentrated by ultrafiltration at 4'C to a volume of 100 pl and the entire content was added to a well. The reference sera in the lateral wells were diluted 1:30 in HBSS and 25 pl was placed in these wells. Diffusion was allowed to proceed for 10- 18 hours at 4°C in a humid chamber.


injections of SRBC at 5 and 10 days and the spleens were assayed for I@ indirect plaques 13 days after the single injection of RNA-treated ni-PEC (1.9- 2.2 X loH cells) (FIGURE 1 I ) . The RNA donor’s allotype was found in 35% of the plaques in one experiment (b5 RNA+b4 SpC) and in 29% of the plaques in another experiment (b4 RNA+b5 SpC). In the control experiment (b4 RNA+b4 SpC), essentially all the plaques were of the b4 allotype. In all of these experiments, the results obtained by radioautography agreed closely with those obtained by the inhibition method. (The 4 % b5 plaques obtained by the inhibition method are not significant; they represent background levels observed by this technique.) These experiments suggest that at least some of the ni-PEC affected by the 5-day i-RNA settle in the spleen and survive, and furthermore, that these cells respond to an antigenic stimulus by proliferating and differentiat- ing to yield IgG antibody-forming cells. The experiments also provide an interesting model for modification of the immune reactivity of the host by in vitro treatment of his own lymphoid cells.

Injection of R N A Extracts

We next considered the possibility that if sufficient amounts of the RNA extracts were injected intravenously, enough of the RNA might remain intact long enough to interact with lymphoid cells in the blood or lymphoid organs. For this purpose, 5-day i-RNA (4.9-6.8 mg) was injected into nonimmunized rabbits and the lymphoid tissues were assayed 18-20 hours, 3 days, and 5 days later for IgM direct plaques.?3.?4 In two typical experiments, in which the analysis was done after 5 days (b5 RNA+b4 SpC and b4 RNA+bS SpC, FIGURE 12), the RNA donor’s light-chain allotype was found in 30% and 32% of the plaques by radioautography. In control experiments (b4 RNA-*b4 SpC and b5 RNA+b5 SpC, FIGURE 13), all of the plaques had the allotype of the host (the few plaques which had the allelic allotype are not considered significant, because of limitations in the methods). The results shown here after 5 days were similar to those observed at 18-20 hours and at 3 days. Moreover, the allotype of the RNA donor was found in the IgG of lymphoid cell lysates as well as in the IgG that was isolated and concentrated from ?4 These results indicate that the RNA when injected can indeed interact with the host’s lymphoid cells to synthesize IgM antibodies and IgG with the allelic allotype.

Injection of R N A Extracts Followed by Multiple Challenge with SRBC

The in vivo system permitted us to ascertain whether the rabbits which have been injected with these RNA extracts respond to subsequent challenge with multiple injections of SRBC to generate IgM and IgG antibodies with the RNA donor’s allotype. The persistence of this response was examined after 13, 21, and 37 days, thereby providing an indication of the in vivo viability of the RNA-affected lymphoid cells and showing whether or not they proliferate and differentiate. Two sets of typical experiments with 5-day i-RNA (FIGURES 14 and 15) and ni-RNA (FIGURES 16 and 17) are illustrated: the rabbits were given SRBC injections at 3 hours and on days 6, 13, 20, 27, and 34 after the RNA injection and the spleens were assayed for IgG indirect plaques after 37 days. The total number of indirect plaques are five- to sixfold greater at 37

212

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FIGURES 10 and 11. Percentage of IgM direct (FIGURE 10) and IgG indirect (FIG- URE 1 1 ) plaques bearing the RNA donor and SpC host allotypes among spleen cells obtained from rabbits after a single intravenous injection of the rabbits' own peri-


days than at 5 d a ~ s . 2 ~ By radioautography, in the experiments with 5-day i-RNA (FIGURE 14, b5 RNA+b4 SpC and b4 RNA-+b5 SpC) , 3 1 % and 30% of the plaques respectively were of the R N A donor's light-chain allotype. In control experiments (FIGURE 15), all of the plaques were of the SpC host's allotype (again, the amounts of the allelic allotype shown by inhibition are not significant). The experiments in which ni-RNA was used gave similar results (FIGURES 16 and 17) .

Finally, the lymphoid cells lysates, the serum, and the IgG isolated and concentrated from the serum were assayed by radio-immunodiffusion for the light-chain allotypes. The results obtained for the experiments shown in FIGURE 16 with ni-RNA show that the R N A donor's allotype could be detected in the lymphoid cell lysates ( L N C and SpC) as well as in the IgG isolated from serum and concentrated (left lower diagrams of FIGURES 18 and 19). The R N A donor's allotype could not be detected in the whole serum by this method. These results indicate that the cells which produce IgM and IgG antibodies with the R N A donor's allotype must result from a proliferation and differentiation of the cells affected by the RNA.

DISCUSSION AND SUMMARY

The use of genetic markers has made it possible to demonstrate clearly that lymphoid cells of one rabbit, if treated in vitro or in v i v o with lymphoid R N A extracted from a rabbit of different genotype with respect to the light and heavy chains of immunoglobulin, are converted to the synthesis of specific IgM and IgG antibody of the R N A donor's light- and heavy-chain allotype. In our

toneal exudate cells (PEC) treated in vifro with 5-day i-RNA. Three rabbits, A76, F15-3, and A34 (FIGURE lo ) , were given a total of 5.5 mg b5-RNA per 2.5 x 10" b4- PEC, 5.1 mg b4-RNA per 1.7 x lo" b5-PEC, and 5.9 mg b4-RNA per 1.9 x lo* b4-PEC respectively. Three rabbits, A49, F15-4, and A45 (FIGURE 1 1 ) were given a total of 6.2 mg b5-RNA per 2 . 2 ~ 10" b4-PEC, 6.5 mg b4-RNA per 1 . 9 ~ 10' b5-PEC, and 5.8 mg b4-RNA per 2.1 x 10" b4-PEC. The PEC were treated with RNA in aliquots (550 pg b5-RNA per 2 . 5 ~ 10' b4-PEC. 510 pg b4-RNA per 1 . 7 ~ 10' b5-PEC, and 590 pg b4-RNA per 1 . 9 ~ 10' b4-PEC in FIGURE 10; 620 pg b5-RNA per 2 . 2 ~ lo' bCPEC, 650 pg b4-RNA per 1.9 x 10' b5-PEC, and 580 pg b4-RNA per 2.1 x lo' b4- PEC in FIGURE 1 1 ) for 30 min at 37°C in a shaking water bath. In all of these experiments, the samples belonging to the same rabbit were then pooled and incubated for an additional 4 hours in a CO? incubator at 3 7 T , in 2 ml HBSS which contained lo' SRBC. The RNA-treated PEC were injected intravenously into nonimmunized rabbits homozygous for the allelic (first two experiments in FIGURES 10 and 11) or the same (last experiment in FIGURES 10 and 1 1 ) light-chain allotype. The spleens of rabbits A76, F15-3, and A34 (FIGURE 10) were assayed for direct plaques after 5 days. Rabbits A49, F15-4, and A45 (FIGURE 11) were given two intravenous injections of 8x lo" SRBC at 5 and 10 days, and then the spleens were assayed for indirect plaques at 13 days. The allotypes of the b kappa light-chain locus in the IgM direct and the I g G indirect plaques were identified (1) by inhibition of plaque formation in the presence of antiallotype antibodies incorporated into the agarose mixture (clear bars), and (2) by radioautography with IgG isolated from anti-b4 and anti-b5 antisera and labeled with "I (shaded bars). Each vertical bar shows the average of quadruplicate determinations. Each of the three experiments in FIGURES 10 and 1 1 was done with a single PEC population obtained from one rabbit and treated with a single RNA preparation. The total number of direct (FIGURE 10) and indirect (FIGURE 1 1 ) plaques per lo" viable SpC is indicated for each experiment.


a

u 40

u K W a

TOTA L=92

b 5 b 4

12

TOTAL = 9 3

b 4 b5 b5RNA- W S p C WRNA-b5SpC

lTAL=87

b4 b 5 b 5 b 4 b4RNA-b4SpC b5RNA- b5SpC


TABLE 2

THAT POSSESS THE RNA DONOR A N D SPC HOST LIGHT-CHAIN AND HEAVY-CHAIN

SUMMARY OF DATA OBTAINED BY RADIOAUTOGRAPHY AND INHIBITION OF PLAQUE FORMATION

PERCENTAGE OF IcM DIRECT AND OF IGG INDIRECT ANTI-SRBC PLAQUES

ALLOTYPES WHEN NI-SPC ARE TREATED WITH 5-DAY AND 24-DAY I-RNA:

Radioautography Plaque Inhibition

Chain % % % % IgM IgG IgM I&

RNA donor light 77-100 64-84 Obs. N C * 67-82 RNA donor heavy 80-86 46-61 75-80 43-62

Host SpC light 7-14 4-3 2 O t 7-28 Host SpC heavy 4-12 30-39 2-1 0 24-3 1

* Obs. NC=observed but not counted. t O=None observed.

studies, SRBC was used as the antigen and the allotypes (genetic markers) of the IgM and IgG antibody plaques formed by SpC were identified with antiallotype antibodies by radioautography and inhibition methods.

Our in vitro data are summarized in TABLE 2. The RNA donor's heavy- chain allotype was found in 75-86% of the IgM plaques and in 43-62% of the IgG plaques. The R N A donor's light-chain allotype was found in 77-100% of the IgM plaques and 64-84% of the IgG plaques. The conversion with respect to heavy-chain allotypes was somewhat less efficient than with respect to light- chain allotypes, presumably because the specific molecules in the R N A extracts which are effective in the synthesis of heavy chains are not present a t optimal concentration.

Our in vivo data are summarized in TABLES 3 and 4. In one set of in vivo experiments, 5-day i-RNA was injected into nonimmunized rabbits and the spleens were assayed at 18-20 hours, 3 days, o r 5 days for the allotype of the IgM plaques (TABLE 3 ) . The R N A donor's light-chain allotype was found in 28-33% of the IgM plaques. In another set of in vivo experiments, 5 d a y

~~ ~ ~ ~~

FIGURES 12 and 13. Percentage of IgM direct plaques bearing the RNA donor and SpC host allotypes among spleen cells obtained 5 days after a single intravenous injection of a 5-day I-RNA extract. The four preparations of 5-day I-RNA (extracted from spleen and lymph nodes) were obtained from b5 homozygous rabbits (F295-4 in FIGURE 12; F357-1 in FIGURE 13) and from b4 homozygous rabbits (BB35 in FIGURE 12; BB30 in FIGURE 13), 5 days after a single i.v. injection of 8 x 10" SRBC." One ml of the RNA extracts (6.0 mg/ml b5-RNA and 5.5 mg/ml b4-RNA in FIGURE 12; 5.8 mg/ml b4-RNA and 5.3 mg/ml b5-RNA in FIGURE 13) were injected into nonimmunized rabbits homozygous for the allelic allotype b4 (BB41) or b5 (G260-2) (FIGURE 12). or for the same allotype b4 (BB40) or b5 (AG323-1) (FIGURE 13)." The spleens were assayed 5 days later for IgM direct plaques bearing the b4 and b5 allotypes by inhibition (clear bars) and radioautography (shaded bars) with anti-b4 and anti-b5. Each vertical bar shows the average of quadruplicate determinations. Each of the two experiments in FIGURES 12 and 13 was done with a single SpC population obtained from one rabbit. The total number of direct plaques per loe viable SpC is indicated for each experiment.


14

'OTAL.980 TOTAL.1520

b 5 b 4 b 4 b 5 b 5 R N A t b 4 S p C b 4 R N A c b 5 SpC

b 4 b 5

15

AL= 523

n b5 b 4

b 4 RNA -b 4SpC 65RNA -b5 SpC

Bell & Dray: Lymphoid Cells

80- cn W 3 0

- 3 n 60-

I- V W - rr n z 40- a J

E -

20- I- z W V - w a a

0-

217

16

TOTAL.28 I

TOTAL = 798

b5 b4 b4 b5 b5RNA -b4SpC WRNAMbSpC

TAL=II2I

17

TOT A L =594

b 4 b5 b 5 b 4 b4RNA-brlSpC b5RNA-b5SpC


FIGURES 14-17. Percentage of IgG indirect plaques bearing the RNA donor and SpC host allotypes among spleen cells obtained from rabbits 37 days after a single intravenous injection of 5-day i-RNA (FIGURES 14 and 15) or ni-RNA (FIGURES 16 and 17) followed by i.v. injections of 8 x 108 SRBC 3 hours later and at days 6, 13, 20, 27, and 34.% Before injection, the RNA preparations (5.3-7.1 mg in 1.0-1.2 ml HBSS) were adjusted to 0.7 M sucrose and contained 8 pg epinephrine and 100 pg DEAE-dextran. The recipient rabbits were homozygous for the alternate allotype (FIGURES 14 and 16) or for the same allotype (FIGURES 15 and 17). The spleens were assayed for IgG indirect plaques bearing the b4 and b5 allotypes by inhibition (clear bars) and radioautography (shaded bars) with anti-b4 and anti-b5. Each vertical bar shows the average of quadruplicate determinations. Each of the two experiments in FIGURES 14-17 was done with a single SpC population from one rabbit. The total number of indirect plaques per lo" viable SpC is indicated for each experiment,

FIGURES 18 and 19. Identification by radioimmunodiffusion of the kappa light- chain allotypes of IgG in whole serum, in isolated IgG preparations from serum, in lymph node cell (LNC) lysates, and in spleen cell (SpC) lysates obtained from a b4 (FIGURE 18) or b5 (FIGURE 19) homozygous rabbit, after a single intravenous injection of 5.8 mg b5 ni-RNA (FIGURE 18) or 6.8 mg b4 ni-RNA (FIGURE 19) followed by an i.v. injection of 8 x 10" SRBC 3 hours later and 5 subsequent injections of SRBC at days 6, 13, 20, 27, and 34 after the RNA injection. The concentration of the IgG from the b4 rabbit was 69 mg/ml (before) and 83 mg/ml (after) in the left diagrams of FIGURE 18. The concentration of the IgG from the b5 rabbit was 71 mg/ml (before) and 80 mg/ml (after) in the left diagrams of FIGURE 19. These IgG preparations were diluted when used in the wells of the right diagrams of FIGURES 18 and 19. The cell lysates in FIGURE 18 were derived from 6.5 x 10' SpC and 7.3 x 10" LNC; in FIGURE 19, they were derived from 6.1 x 10' SpC and' 6.9 x 10" LNC. The allotypes were identified by double diffusion in 1.0% agarose, which contained 0.75% deoxycholate in 0.1 M sodium borate buffer at pH 8.6, with '9 IgG-labeled anti-b4 and anti-b5 placed in the center wells. The reference b4(*) (at a 1: 100 dilution) and b5(*) (at a 1:85 dilution) sera from homozygous rabbits were placed in the lateral wells.



TABLE 3 PERCENTAGE OF IGM DIRECT ANTI-SRBC PLAQUES THAT POSSESS THE RNA DONOR

OR 5 DAYS AFTER THE INJECTION OF 5-DAY I-RNA: SUMMARY OF DATA OBTAINED BY RADIOAUTOGRAPHY AND INHIBITION OF PLAQUE FORMATION

AND SPC HOST LIGHT-CHAIN ALLOTYPES IN SPC OF RABBITS, 18-20 HOURS, 3 DAYS,

Radioautography Plaque Inhibition IgM IgM

Chain % %

RNA donor light 29-32 28-33 Host SpC light 47-6 1 45-64

i-RNA or ni-RNA was injected into nonimmunized rabbits and was followed by multiple injections of SRBC to evoke an IgG response (TABLE 4). After 13, 21, and 37 days, the RNA donor’s lightchain allotype was detected in 2542% and the RNA donor’s heavy-chain allotype was found in 12-19% of the IgG plaques.

Several investigators have reported that i-RNA-treated lymphoid cells will synthesize the specific antibody being made by the RNA c9. 33-30 The probable contamination of the RNA extracts with antigen has evoked consider- able effort to evaluate the possibility that an RNA-antigen complex acts as a superantigen to induce antibody formation.ln, 12-14, 33-37 Contamination with antigen, however, can hardly explain the appearance of the RNA donor’s light- and heavy-chain allotypes.4~ l0-z5 Passive immunization of the recipient ni-SpC as a result of possible contamination of the RNA extracts with antibody has also been considered. By quantitative studies in vitro, we have shown that a net synthesis of IgM and IgG antibody occurs when ni-SpC are treated with 1-RNA (FIGURE 4; see also Bell and Dray ? O ) . This is also indicated by the work of Walker and c01leagues.S~

In addition to assessing the allotypes of specific antibody molecules, we have

TABLE 4

THE RNA DONOR AND SPC HOST LIGHT-CHAIN AND HEAVY-CHAIN ALLOTYPES IN SPC PERCENTAGE OF IGM DIRECT AND IGG INDIRECT ANTI-SRBC PLAQUES THAT POSSESS

OF RABBITS, 13, 21, OR 37 DAYS AFTER THE INJECTION OF S-DAY I-RNA OR NI-RNA FOLLOWED BY MULTIPLE INJECTIONS OF SRBC: SUMMARY OF DATA OBTAINED

BY RADIOAUTOGRAPHY AND INHIBITION OF PLAQUE FORMATION.

Radioautography Plaque Inhibition IgM IgG IgM IgG

Chain % % % %

RNA donor light 22-30 25-3 3 23-32 28-42 RNA donor heavy 12-18 15-19 10-19 12-19

Host SpC light 56-67 57-66 59-65 58-7 1 Host SpC heavy 51-57 55-60 55-65 59-69


also assayed, by immunodiffusion, the lysates of the RNA-treated lymphoid cells for the appearance of IgG molecules with the RNA donor’s allotype. In the in vivo experiments, the serum was also assayed. The RNA donor’s light- and heavy-chain allotypes were identified in the lysates of RNA-treated SpC suspensions. The quantitative relationships in the in vitro experiments clearly indicated that the RNA-treated SpC actively synthesized the RNA donor’s allotype (FIGURE 9) and that at least 7-10 pg of the RNA donor’s allotype was synthesized by 2.2 X loq cells after 4.5 hours of incubation with 470 pg RNA. The RNA donor’s allotype was not found in the controls with the same amount of the RNA or with lysates derived from the same amount of untreated SpC.

In the in vivo experiments, the RNA donor’s allotype was also found in the IgG isolated from serum after 18-20 hours, 3 days, and 5 days in one set of experiments and after 13, 20, and 37 days in another set of experiments. The percentage of the RNA donor’s allotype in serum was very small, however, less than 1 %, and did not reflect the percentage of the RNA donor’s allotype that was found among the anti-SRBC antibody population. Perhaps sufficient time did not elapse for the bulk IgG to reflect the distribution among the plaque- forming cells, or perhaps an immune response to the RNA donor’s allotype may result in the active removal of the foreign allotype immediately after synthesis.

The RNA extracts used in our experiments were inactivated by treatment with RNase but not by DNase or by trypsin.’!l-z5 Thus it appears that the RNA-mediated conversion of lymphoid cells was due to the transfer of information contained in the base sequence of RNA. Since transfer has occurred with respect to both light and heavy chains of immunoglobulins, this would imply the function of at least two such RNA molecules. One possibility is that the RNA acts as a direct template for protein synthesis. This seems to be ruled out by the in vivo experiments which indicated the continued and increased magnitude of the response after 37 days, reflecting not only survival of the RNA- treated cells but also their proliferation and differentiation. It therefore seems more likely that the genetic information of the foreign RNA is stabilized in the host, e.g., by a de novo synthesis of DNA through the activity of an RNA- dependent DNA polymerase system, as has been found in mammalian cells infected with RNA viruses.3!!’. ‘‘I It is also possible, however, that the structural genes for the b4 and b5 polypeptide chains are not truly allelic. Thus, although extensive genetic data are available which are consistent with allelism,’s~ ‘ I , J2

the DNA for the b4 and b5 polypeptide chains may indeed be present in both b4 and b5 homozygous rabbits and the allelic genes which control the synthesis of these polypeptide chains may be control rather than structural genes; and they may act by allowing either the b4 or b5 DNA chromosomal regions to be expressed. If this were the RNA extracts might be eliminating or modifying the action of the control or regulatory genes and thus leading to the results we have obtained. Indeed, Rivat and colleagues43 have reported that the IgG immunoglobulins produced by human lymphocytes which are cultured in the presence of phytohemagglutinin may possess Gm allotypes not found in serum. They suggested that the segregation of regulatory genes rather than of structural genes may account for the patterns of inheritance of the G m allotypes.4:3 Nevertheless, Cohn ‘’ has considered this latter explanation unlikely on theoreti- cal grounds. Finally, we consider it unlikely from the fact that the allelic light- or heavy-chain allotypes do not appear in allotype-suppressed rabbits homozygous for the a heavy-chain or h light-chain locus; rather, light chains of an unlinked locus and heavy chains of a closely-linked locus appear instead.45-4R


ACKNOWLEDGMENTS

We are grateful to Nancy Biskup and to Mei Cheng for skillful technical assistance and to Alice Gilman-Sachs for characterizing the antiallotype sera. We thank Doctor Leon LeBeau for aid in photography and Doctor Katherine Knight for a generous supply of goat anti-rabbit Fc IgG.

REFERENCES

1. FISHMAN, M. 1961. J. Exp. Med. 114: 837. 2. FISHMAN, M. & F. L. ADLER. 1963. J. Exp. Med. 11% 595. 3. FISHMAN, M. & F. L. ADLER. 1963. Third International Immunopathology

Symposium. La Jolla, California. P. Grabar & P. Miescher, Eds. : 79. Grune & Stratton, Inc. New York, N.Y.

4. ADLER, F. L., M. FISHMAN & S. DRAY. 1966. J, Immunol. 97: 554. 5. COHEN, E. P. & J. J. PARKS. 1964. Science 144: 1012. 6. COHEN, E. P., R. W. NEWCOMB & L. K. CRoseY. 1965. J. Immunol. 94: 416. 7. &HEN, E. P. 1967. Nature 213: 462. 8. FRIEDMAN, H. 1964. Science 146: 934. 9. FRIEDMAN, H. 1963. Nature 199: 502.

10. FRIEDMAN, H. P., A. B. STAVITSKY & J. M. SOLOMON. 1965. Science 149: 1106. 11. JERNE, N. K., A. A. NORDIN & C. HENRY. 1963, In Conference on Cell Bound

Antibodies. B. Amos & H. Koprowski, Eds. : 109. Wistar Institute Press. Philadelphia, Pa.

12. ASKONAS, B. A. & J. M. RHODES. 1965. Nature 205: 470. 13. GOTTLIEB, A. A. 1968. I n Nucleic Acids in Immunology. 0. J . Plescia &

W. Braun, Eds. : 471. Springer-Verlag New York Inc. New York, N.Y. 14. GOTTLIEB, A. A. 1968. J. Reticuloendothel. SOC. 5: 270. 15. ASKONAS, B. A. & J. M. RHODES. 1965. In Molecular and Cellular Basis of An-

tibody Formation. J. Sterzl, Ed. : 503. Academic Press Inc. New York, N.Y. 16. MERRITT, K. &A. G. JOHNSON. 1965. J. Immunol. 94: 416. 17. BRAUN, W. 1965. I n Molecular and Cellular Basis of Antibody Formation :

18. BRAUN, W. & E. P. COHEN. 1968. Regulation of the Antibody Response.

19. BELL, C. & S. DRAY. 1969. J. Imrnunol. 103: 1196. 20. BELL, C. & S. DRAY. 1970. J. Immunol. 105: 541. 21. DRAY, S. & C. BELL. 1970. Immunochemistry 7: 856 (Abstr.). 22. BELL, C. & S. DRAY. 1971. J. Immunol. 107: 83. 23. BELL, C. & S. DRAY. 1971. Science 171: 199. 24. BELL, C. & S. DRAY. 1972. Cell. Immunol. 5: 52. 25. BELL, C. & S. DRAY. 1973. Cell. Immunol. In press. 26. STERZL, J. & I. RIHA. 1965. Nature (London) 208: 858. 27. WORTIS, H. H., R. B. TAYLOR & D. W. DRESSER. 1966. Immunology 11: 603. 28. DRAY, S., G . 0. YOUNG & L. GERALD. 1963. J. Immunol. 91: 403. 29. UYEKI, E. M. & R. S. KLASSEN. 1968. J. Immunol. 101: 271. 30. SCHERRER, K. & J. E. DARNELL. 1962. Biochem. Biophys. Res. Cornmun. 7:

31. THOR, D. E. & S. DRAY. 1968. J. Immunol. 101: 469. 32. HELMREICH, E., M. KERN & H. N. EISEN. 1961. J. Biol. Chem. 236: 464. 33. MITSUHASHI, S., K. SAITO, N. OSAWA & S. KURASHIGE. 1967. J. Bacteriol. 94:

34. KURASHIGE, S., K. KITAMURA, K. AKAMA & S. MITSUHASHI. 1970. Japan. J.

525. Academic Press Inc. New York, N.Y.

Charles C Thomas, Publisher. Springfield, Ill.

486.

907.

Microbiol. 14: 41.


35. YAMAGUCHI, N., S. KURASHIGE & S. MITSUHASHI. 1971. J. Immunol. 107: 99. 36. JACHERTZ, D. & J. DRESCHER. 1970. J. Immunol. 104: 746. 37. FISHMAN, M. & F. L. ADLER. 1967. Cold Spring Harbor Symp. Quant. Biol.

38. WALKER, W. S., M. FISHMAN & F. L. ADLER. 1971. J. Immunol. 107: 953. 39. BALTIMORE, D. 1970. Nature 226 1209. 40. TEMIN, H. M. & S. MIZUTANI. 1970. Nature 226: 1211. 41. OUDIN, J. 1960. J. Exp. Med. 112: 107, 125. 42. DUBISKI, S., A. DUBISKA, D. SKALBA & A. KELUS. 1961. Immunology 4: 236. 43. RIVAT. L., D. GILBERT & C. ROPARTZ. 1970. Immunology 19: 959. 44. COHN, M. 1971. Ann. N.Y. Acad. Sci. 190: 529. 45. VICE, J. L., W. L. HUNT & S. DRAY. 1969. J. Immunol. 103: 629. 46. VICE, J. L., A. GILMAN-SACHS, W. L. HUNT & S. DRAY. 1970. J. Immunol. 104:

47. KNIGHT, K. L., A. GILMAN-SACHS, R. FIELDS & S. DRAY. 1971. J. Immunol.

48. KIM, B. S. & S. DRAY. 1973. European J. Immunol. In press.

32: 343.

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106: 761.

DISCUSSION OF THE PAPER

DR. RIGBY (Creighton Medical College, Omaha, Nebr . ) : Do you have information on the efficiency of your R N A delivery system? How much of the R N A that you've made is actually delivered and is effective, either in vitro or in vivo? Did you alter the delivery system in terms of such characteristics as temperature, time of incubation, the use of other materials to increase the efficiency of the system, and so on?

DR. BELL: We d o not know how much of the R N A is actually entering the cells and how much of this R N A present in the cell is necessary for the conversion phenomena to occur. When we titrated the amount of R N A that is necessary for obtaining P F C in our in vitro system, we obtained in vitro conversion with as little a s 50 pg with some preparations of RNA, while 500 pg R N A was needed with other preparations. The amount of total R N A used in these systems varied from preparation to preparation. Incubated with i-RNA for 5-15 min at 37" C in vitro, ni-SpC will convert to PFC, but no conversion appeared to occur when the ni-SpC were incubated at 4°C even after 1-2 h r of incubation. When the delivery system was altered, e.g., preincubation of cells with either epinephrine or a combination of 100 pg DEAE-dextran and 0.3 M sucrose prior to R N A treatment, a higher conversion seemed to occur both in terms of the number of PFC and in terms of allotype conversion. This might indicate that the permeability of the cell membrane changes t o the extent that either: 1 ) more R N A is entering the cells, thus leading to a higher synthesis of protein; or 2 ) the synthesis of protein remains constant, but more hemolysins (antibody) are secreted through the cell.

DR. R. H. SCHWARTZ (Rutgers State University, New Brunswick, N . J . ) : What d o you think is the cell source of your immune RNA?

DR. BELL: We d o not know the exact cellular source. In most of our experiments we have used RNA extracted either from lymph nodes or from the spleen. In earlier experiments we noted that peritoneal exudate cells from immune rabbits were as effective in inducing a direct IgM response as the R N A extracted from the lymph nodes. We also did some in vitro experiments with RNA from bone marrow, thymus, liver, kidney, heart, appendix, and brain.


The effectiveness of the RNA seemed to correlate with the content of cells from the reticuloendothelial system.

It is not clear to me why any cells in the recipient would make the allotype of their own. Does this point to some antigenic contamination in the RNA preparation? In this regard, have you on any occasion simultaneously given the immunogenic RNA and antigen?

DR. BELL: We probably have a residual amount of antigen that does induce an immune response in the recipient animals. Treated with 5-day-i-RNA, ni-SpC convert to IgM PFC, 5-10% of which bear the host allotype. Treated with 24-day-i-RNA, ni-SpC convert to IgG PFC about 30% of which bear the host allotype. This might suggest that the residual antigen is present in higher concentration in 24-day-i-RNA. When the in vitro experiments were performed in the presence of i-RNA and antigen, the number of PFC was slightly higher. However, the allotype content remained essentially unchanged.

DR. COHEN: Is there any evidence to show that those cells of the recipient which respond to the RNA extracts are the same as those which would respond to direct immunization?

DR. BELL: No. DR. ADLER (Public Health Research Institute of the City of New York,

Znc., New York, N . Y . ) : It seems that the Chicago group along with Dr. Fishman and myself are about the only people who utilize the rabbit allotype system. We would welcome company in this field. Secondly, Dr. Haurowitz, in his initial paper of this meeting, raised the fundamental question: Where do various bits of information-such as, those from the DNA level, the RNA level, and the peptide chains-go into the antibody molecule? Your very beautiful experiments indicate that the normal RNA which carries allotype information goes into a recipient rabbit that lacks this allotype; this is followed by a second manipulation which provokes this rabbit into making antibody of that allotype. Thus, these two bits of input combine to make the specific response. I believe this helps us well along toward answering the question raised by Dr. Haurowitz. DR. H. FRIEDMAN (Albert Einstein Medical Center, Philadelphia, Pa.) :

When you treat your tissue cultures with immunogenic RNA, do your target cells undergo division? Is division required for the expression of antibody synthesis, and is RNA and protein synthesis necessary for the response? Also, what is the size of your active RNA preparation in terms of molecular weight?

DR. BELL: We do not know whether the target cell has to divide in response to RNA treatment in vitro in order to observe plaques to SRBC. The variation in the number of cells cultured in vitro does not seem significant; however, protein synthesis must occur in order to observe PFC to SRBC. Thus, when ni-SpC were preincubated with 50 pg puromycin prior to RNA treatment in the presence of 1+C leucine, both protein synthesis and plaque formation were inhibited. We do not know to what extent de novo RNA synthesis is necessary for the immune response observed. With respect to the molecular size of RNA, the sucrose density gradient pattern exhibited approximately twice as much 28s RNA as 18s RNA and relatively smaller amounts of 4-5 S RNA. When either the 28s or 18s peak were slightly degraded, the RNA seemed inactive in converting ni-SpC to PFC.

DR. E . P. COHEN (University of Chicago, Chicago, Illinois):

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LYMPHOID CELLS CONVERTED BY LYMPHOID RNA EXTRACTS IN VITRO AND IN VIVO TO SYNTHESIZE ALLOGENEIC...

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